Characterization of monoclonal anti-human factor VII antibody (B101/B1) that recognized three-dimensional structures near Arg at position 79 in the first EGF-like domain.
Murine monoclonal antibodies (designated hVII-B101/B1, hVII-DC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVII-DC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10(-10) M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced gamma-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.
Takamiya
O
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Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, Asahi, Japan.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000097:blood*; D000937:Antigen-Antibody Reactions / Q000187:drug effects*; D001120:Arginine; D002118:Calcium / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000737:chemistry*; D000939:Epitopes; D005167:Factor VII / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004815:Epidermal Growth Factor; D005167:Factor VII; D001120:Arginine; D002118:Calcium